We would like to completely define the nucleotide sequence of a viral DNA, and then to understand the functional significance of each nucleotide in this sequence. In order to understand the sequence we plan to develop techniques for altering it at will by chemical and enzymatic procedures. We also proposed to develop general and practical methods for the construction of mutants containing any desired alteration of known DNA sequence. We hope to apply such methods to construct mutants in regions of the PhiX174 genome where sequence is known but genetic function is still a mystery. We hope to apply our methods for the construction of altered DNA sequences to the analysis of other bacterial genetic material, and eventually to eukaryotic genetic elements propagated in bacteria. During the coming year we plan: (1) To produce a family of PhiX174 gene E ribosome binding site mutants. (2) To determine the exact point (with respect to its CCNGG recognition site) at which Sa1II cleaves DNA. (3) To apply the DNA sequence library approach to other protein-DNA interactions. (4) To begin the application of site directed mutagenic techniques to mouse beta-globin genes cloned in M13 phage vectors.